In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wildtype B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.GPCR 1 constitute the largest family of receptors and mediate responses to numerous agonists including hormones, neurotransmitters, and sensory stimuli. Receptor activation by the agonist ligand is considered the first step in receptor signal transduction. However, the recent discovery that GPCR exhibit ligand-independent, spontaneous, and mutation-induced activity has introduced a new function that needs to be considered in cellular signaling and ligand action (1) and that can lead to disease (2-4).GPCR have evolved as the most common targets for therapeutic drugs (5). Drug efficacy is defined as the capacity of a drug to activate or inactivate a receptor response and is fundamental to pharmacology and drug development. Classically, this parameter has been considered an intrinsic property of the drug and independent of the cellular environment in which the drug acts (6 -8). Partial agonists, or mixed agonists/antagonists, are prevalent among GPCR drugs and are defined as ligands that elicit a submaximal receptor response and block the response to the natural receptor ligand, a full agonist, or any other ligand of higher efficacy. It has been predicted that in the absence of a more efficacious agonist, the efficacy and behavior of a partial agonist should be directly dependent upon the level of spontaneous receptor activity (9). In other words, if the partial agonist ligand elicits a response that is higher than the spontaneous activity, then the ligand should behave as an agonist, whereas if the ligand elicits a lower response, then the ligand should block t...

In order to identify agonist-and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and LeebLundberg, L. M. Seven-transmembrane domain GPCR 1 constitute by far the largest family of plasma membrane receptors. These receptors bind ligands of widely diverse origins, and are unsurpassed as therapeutic targets. Consequently, much effort has been devoted to mapping of the binding sites for agonist and antagonist ligands in these receptors (1, 2). Even though peptides are the most common class of ligands for GPCR, few peptide GPCR have been investigated thus far, and in most of those cases the identity of the peptide-binding epitopes remains elusive.Receptors for kinins, pro-inflammatory peptides 8 -10 amino acids in length, have been classified into two subtypes, termed B1 and B2 (3), and are members of the GPCR superfamily (4, 5). These receptor subtypes, although only 36% identical, discriminate between peptide agonists that differ only in their C-terminal residue; BK binds to the B2 receptor, whereas the C-terminally truncated carboxypeptidase fragments desArg 9 -BK and des-Arg 10 -Lys-BK, or des-Arg 10 -KD, bind to the B1 receptor. Several high affinity B2 receptor-selective decapeptide antagonists structurally derived from BK have been developed, including NPC17731 and HOE140 (6 -8). Interestingly, the fact that the des-Arg 10 analogs of these peptides act as high affinity B1 receptor-selective antagonists emphasizes the significance of the C-terminal Arg in receptor subtype selectivity (9 -11).In the B2 receptor, extensive analysis of most of the TMs and a significant amount of the ECs by alanine-scanning mutagenesis has yielded no information about residues important for antagonist binding and has identified only a few residues important for agonist binding (12-15). We recently developed a novel, potentially more effective strategy for mapping the binding sites in kinin receptors, which is based on the identification of receptor epitopes that enable these receptors to discriminate between ligands (11). This strategy involves the exchange of individual TMs between the B1 and B2 receptor subtypes and the subsequent exchange of non-conserved residues that are possible candidates for discriminatory action. This approach is intrinsically more reliable than alanine-scanning mutagenesis as it yields in sequence both loss-of-function and gain-of-function mutations.In an initial study, we used this strategy to identify specific residues in TM-VI of the human WT B1 and B2 receptors that are partially responsible for enabling these receptor subtypes to discriminate between peptide agonists (11). In the present study, we analyzed the role of TM-III in peptide ligand discrimi...

The B1 bradykinin (BK) receptor (B1R) is a seventransmembrane domain, G protein-coupled receptor that is induced by injury and important in inflammation and nociception. Here, we show that the human B1R exhibits a high level of ligand-independent, constitutive activity. Constitutive activity was identified by the increase in basal cellular phosphoinositide hydrolysis as a function of the density of the receptors in transiently transfected HEK293 cells. Several B1R peptide antagonists were neutral antagonists or very weakly efficacious inverse agonists. Constitutive B1R activity was further increased by alanine mutation of Asn 121 in the third transmembrane domain of the receptor (B1A 121 ). This mutant resembled the agonist-preferred receptor state since it also exhibited increased agonist affinity and decreased agonist responsiveness. A dramatic loss of constitutive activity occurred when the fourth intracellular C-terminal domain (IC-IV) of the human B2 BK receptor subtype (B2R), which exhibits minimal constitutive activity, was substituted in either B1R or B1A 121 to make B1(B2ICIV) and B1(B2ICIV)A 121 , respectively. Activity was partially recovered by subsequent alanine mutation of a cluster of two serines and two threonines in IC-IV of either B1(B2ICIV) or B1(B2ICIV)A 121 , a cluster that is important for B2R desensitization. The ligand-independent, constitutive activity of B1R therefore depends on epitopes in both transmembrane and intracellular domains. We propose that the activity is primarily due to the lack of critical epitopes in IC-IV that regulate such activity.The B1 and B2 bradykinin (BK) 1 receptors are seven-transmembrane domain, G protein-coupled receptors (GPCR) (1, 2), which mediate the actions of kinins (3), pro-inflammatory peptides formed in response to tissue injury from kininogen precursors (4, 5). Kinin actions include pain, inflammation, and hyperalgesia (4, 6). The B2 receptor mediates the actions of BK and Lys-BK or kallidin (KD), the first set of bioactive kinins formed following injury, whereas the B1 receptor is thought to mediate the actions of des-Arg 9 -BK and des-Arg 10 -KD, the carboxypeptidase products of BK and KD and the second set of bioactive kinins formed (3). Despite binding very similar ligands, leading to their classification as receptor subtypes, these receptors exhibit only 36% identity. Furthermore, the B2 receptor is constitutively expressed, whereas the B1 receptor is expressed at very low levels, if at all, in healthy tissue but is induced by inflammatory stimuli such as interleukin-1␤ (2, 7-9) and by kinins themselves (8, 10). This pattern of expression is consistent with the fact that the B2 receptor appears to be the principal kinin receptor under healthy conditions and in the acute stage of the inflammatory response (4, 6, 11, 12), whereas the B1 receptor is important primarily in the chronic stage of the response (6, 13, 14).The rationale for restricting B1 receptor expression primarily to conditions of injury is not understood. One explanation is that th...

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N'-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent Kd of about 60 microM. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.